Multiple signaling pathways converge on the Cbfa1/Runx2 transcription factor to regulate osteoblast differentiation

Abstract

The Cbfa1/Runx2 transcription factor is essential for osteoblast differentiation. However, levels of Runx2 are often not well correlated with its transcriptional activity suggesting that this factor must be activated either by covalent modification or through interactions with other nuclear components. Runx2 is phosphorylated and activated by the mitogen-activated protein kinase (MAPK) pathway. This pathway is stimulated in at least two ways: by binding of type I collagen to alpha2beta1 integrins on the osteoblast surface and by treatment of cells with the osteogenic growth factor, FGF2. Protein kinase A (PKA) also may phosphorylate/activate Runx2 under certain conditions. Runx2 activity also is enhanced by factors known to stimulate specific signal transduction pathways such as PTH/PTHrP (signals through PKA and PKC pathways) and BMPs (Signal through Smad proteins). Interactions with Runx2 are complex involving both binding of distinct components such as AP-1 factors and Smads to separate sites on DNA, direct interactions between Runx2 and AP-1/Smad factors and MAPK or PKA-dependent Runx2 phosphorylation. These findings suggest that Runx2 plays a central role in coordinating multiple signals involved in osteoblast differentiation.

Figure 1

Activation of the MAPK pathway stimulates phosphorylation within the P/S/T domain of Runx2. (A) Runx2 domains. Diagram shows the three major domains of Runx2: the glutamine/alanine-rich region (Q/A), the Runt domain that contains DNA binding and nuclear localization sequences (NLS), and the proline/serine/threonine (P/S/T) domains. Inset shows the amino acid sequence of a region of the P/S/T domain shown to be crucial for activation of transcription by the MAPK pathway. Consensus MAPK phosphorylation sites are indicated by asterisks. (B, C) Runx2 phosphorylation. Wild-type Runx2 (Panel B) or a P/S/T domain deletion mutant (ΔPST, Panel C) both containing a N-terminal HA tag were expressed in COS7 cells using a pcDNA5 expression vector. Cells were co-transfected with either pcDNA5 containing a β-galactosidase cDNA (β-gal) or a constitutively active MEK cDNA, MEK(SP). Cells were metabolically labeled with either ³²P orthophosphate or ³⁵S methionine/cysteine, and cell extracts immunoprecipitated with an anti-HA epitope monoclonal antibody. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography.

Figure 2

FGF2-dependent activation and phosphorylation of Runx2. (A) Activation of the OCN promoter. MC3T3-E1 preosteoblast cells stably transfected with a 1.3 kb fragment of the mouse osteocalcin gene 2 promoter driving a firefly luciferase reporter gene were treated with vehicle or 12.5 ng/mL FGF2 for 6 hr in the presence or absence U0126, a MEK inhibitor, or the inactive analogue, U0124. (B) Runx2 phosphorylation. MC3T3-E1 cells were metabolically labeled with ³²P orthophosphate and treated with the indicated concentrations of FGF2 for 6 hr in the presence or absence of U0126 or U0124. Endogenous Runx2 was immunoprecipitated with a polyclonal antibody and analyzed by SDS-PAGE and autoradiography (upper panel). Total Runx2 in immunoprecipitates was measured by Western blotting (lower panel).

Figure 3

Synergistic stimulation of osteoblast gene expression by transduction of mesenchymal cells with adenoviruses overexpressing Runx2 and BMPs. (A) Alkaline phosphatase activity. C3H10T1/2 cells were transduced with optimal titers of adenoviruses containing a LacZ control cDNA (LacZ) or cDNAs encoding BMPs 2, 4, or 7 and/or Runx2 (R) as indicated. Cells were harvested after 6 days and assayed for alkaline phosphatase activity. (B) mRNA levels. Cells were transduced with AdLacZ, AdBmp2, or AdRunx2 adenovirus as indicated. After various times, total RNA was isolated for Northern blot analysis of Runx2, and OCN mRNAs as well as for 18S rRNA for blot normalization.

Figure 4

Overview of signal transduction pathways affecting Runx2 activity. Refer to text for explanation.